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Title:Effect of uptake pathway on non-viral gene delivery in vitro and in vivo
Author(s):Shum, Wing
Director of Research:Pack, Daniel W.
Doctoral Committee Chair(s):Pack, Daniel W.
Doctoral Committee Member(s):Kraft, Mary L.; Yang, Hong; Roy, Edward J.
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Gene delivery
Non-viral
Polyethylenimine (PEI)
endocytosis
xenograft tumor
in vivo gene delivery
clathrin
caveolin
transferrin
folate
receptor-mediated endocytosis
Abstract:Efficient non-viral gene delivery often involves the conjugation of a cell-specific ligand to the vector, which directs the vector to its intended target through binding to a cellular receptor followed by internalization via endocytosis. However, little is known in terms of how the various endocytosis pathways affect the performance of the delivery vehicle. Previously, the Pack lab has demonstrated that caveolin-mediated endocytosis is important to in vitro polyethylenimine (PEI)- and polyamidoamine (PAMAM)-mediated gene delivery in HeLa cells through the use of small molecule drugs and small-interfering RNA [1-2]. The goal of this thesis is to further elucidate the effects of cellular uptake mechanism on non-viral gene delivery in vitro and in vivo utilizing a small hairpin RNA (shRNA) Tet-on system. Specifically, we have investigated the effects of clathrin-dependent endocytosis and caveolin-dependent endocytosis on the efficacy of PEI and PEI-derivative gene delivery in HeLa and MDA-MB-231 cell cultures and xenograft tumors. First, we attached transferrin and folate ligands to PEI to direct internalization by clathrin- and caveolin-mediated mechanisms, respectively, and transfected HeLa and MDA-MB-231 cells with targeted and untargeted PEI in the presence of small molecule drugs that inhibit clathrin- or caveolin-mediated endocytosis. We demonstrated through these studies that caveolin-dependent endocytosis is important to successful PEI-mediated gene delivery in both HeLa and, for the first time, MDA-MB-231 cells. In addition, a similar endocytic pathway study was performed using biodegradable PEI and acetylated PEI, which have demonstrated superior gene delivery capability compared to unmodified PEI in previous work [3-4]. The data show that regardless of structure, size, molecular weight, or zeta potential of the PEI-derivative polyplexes, caveolin-dependent endocytosis was critical to effective gene delivery. In order to further understand the impact of endocytic pathways on gene delivery efficiency in vitro, we modified HeLa and MDA-MB-231 cells to express small hairpin RNA (shRNA) that inhibit expression of clathrin, caveolin, or lamin A/C (negative control) protein expression (HeLa-CLTC, HeLa-CAV, HeLa-LAM, 231-CLTC, 231-CAV, and 231-LAM) in the presence of tetracycline. Using the shRNA Tet-on system, we demonstrated ~90% clathrin knockdown and ~60% caveolin knockdown in both modified HeLa and MDA-MB-231 cells. Subsequently, utilizing this inducible shRNA protein knockdown system, we transfected modified shRNA cells using both targeted and untargeted PEIs and confirmed that caveolin-dependent endocytosis is more important than clathrin-dependent endocytosis to PEI-mediated gene delivery, which corroborates the results observed in unmodified cell lines using small-molecule endocytosis inhibitors. Lastly, we investigated the effect of clathrin-dependent and caveolin-dependent endocytosis on in vivo PEI-mediated gene delivery in a xenograft murine tumor model. By inoculating modified shRNA cells into NIH-III nude mice, we created a xenograft tumor model that allows us to induce CLTC and CAV protein knockdown inside the tumor. Using this Tet inducible system, we demonstrated that the mice fed with Tet-containing water, compared to those fed with water only, show over 50% target protein knockdown in modified MDA-MB-231 cells and 90% CLTC knockdown and 35% CAV knockdown for modified HeLa cells. By transfecting shRNA modified tumors intratumorally with PEI polyplexes, PEI gene delivery efficacy was 15-fold and 2.5-fold higher for modified HeLa and MDA-MB-231 tumors, respectively, when comparing clathrin- versus caveolin-dependent endocytosis inhibition, although the improvement for unmodified MDA-MB-231 tumors was statistically insignificant. The results confirmed that caveolin-dependent endocytosis was more important to in vivo PEI-mediated gene delivery than clathrin-dependent endocytosis, which is in good agreement with in vitro data using both small molecule drugs and Tet inducible protein knockdown. In conclusion, these studies of intracellular trafficking and its effect on PEI gene delivery efficacy in cancer cell lines and tumors will aid researchers in designing more efficient non-viral gene delivery vectors for cancer gene therapy, by synergistically combining the design of cell targeting ligands and the understanding of cellular uptake mechanism for polymeric carriers.
Issue Date:2013-08-22
URI:http://hdl.handle.net/2142/45665
Rights Information:Copyright 2013 Wing Shum
Date Available in IDEALS:2013-08-22
2015-08-22
Date Deposited:2013-08


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